KR102364427B1 - A pesticide composition comprising piperine, and uses thereof - Google Patents

Abstract

The present invention relates to an insecticidal composition comprising piperine or a derivative thereof as an active ingredient to prevent infection of plant parasitic nematodes, a method for controlling plant parasitic nematodes using the same, a method for controlling plant diseases, and a kit comprising the same, The composition according to the present invention contains piperine or a derivative thereof as an active ingredient to not only kill the larvae and adults of the plant parasitic nematode, Bursaphelenchus xylophilus , but also inhibit the differentiation of eggs. Because it appears, it can be provided as an effective insecticidal agent for plant parasitic nematodes and a control or therapeutic agent for plant diseases.

Description

A pesticide composition comprising piperine, and uses thereof

The present invention relates to an insecticidal composition comprising piperine or a derivative thereof and a method for controlling plant diseases using the same.

Pine wilt disease is a plant disease that causes serious damage to forest ecosystems in various regions of Asia, Europe and North America. It was first reported in Busan in 1988 in Korea. Abies), spruce (Picea), and some species of larch (Larix) are also known to occur.

Pine wilt disease is caused by infection with a plant parasitic nematode, Bursaphelenchus xylophilus , which infects a plant. In a tree infected with the pine wilt nematode, resin secretion stops and volatile substances such as alcohol and terpene are secreted. In addition, abnormalities in the flow of water and nutrients cause death, and once infected with the pine wilt nematode, there is hardly any treatment and the tree will die.

The pine wilt nematode ( Bursaphelenchus xylophilus ), which causes pine wilt disease, is taxonomically classified as Metazoa, Eumetazoa, Bilateria, Pseudocoelomata, Nematode (Nematoda, Nemata). It is an animal belonging to the genus and has the shape of a thread. Although the length is less than 1 mm, the fertility is so high that a pair can grow to 200,000 in 20 days. Since the pine wilt nematode cannot invade trees or move between trees by itself, the infection spreads through vectors, and monochamus alternatus or M. saltuarius is known as the main vector.

The route of infection of pine wilt disease is that when the vector lays eggs on a tree infected with the pine wilt nematode, the pine wilt nematodes gather around when the eggs of the vector worm hatch and pupate, and when the vector worm molts, the pine wilt nematode enters the body of the vector worm. It invades, and the moulted vector migrates to a new tree, thereby infecting the new tree with pine wilt nematodes.

Several approaches have been used to control pine wilt disease, such as fumigating wood materials or injecting insecticides into the trunk. The most commonly used insecticides are abamectin and emamectin benzoate, which belong to the avermectin family. Insecticides containing these chemicals are very effective against certain pine wilt nematodes and are considered environmentally safe, but have a disadvantage in that they are expensive. In addition, repeated use of pesticides such as abamectin and evermectin induces selection pressure and develops resistance to the pesticide, as evidenced by several pests such as the Colorado potato beetle, diamondback moth, and nematode nematode. There is a problem that the insecticidal or control effect is weakened.

On the other hand, some studies have reported that natural substances such as phytochemicals, essential oils, and natural esters have an insecticidal action on nematodes, and flavonoids, polyphenols ( Phytochemicals such as polyphenol) are attracting attention in that they selectively kill only pests that are non-toxic to plants and act as pathogens.

The present inventors have completed the present invention by confirming the insecticidal effect of piperine, a kind of phytochemical, in order to provide a formulation of a new natural material that exhibits an insecticidal or control effect on pine wilt nematode in consideration of this point. .

Republic of Korea Patent Publication No. 10-2018-0116627

Rajasekharan, et al. Scientific reports 7.1 (2017): 6803.

The present invention provides an insecticidal composition for preventing infection of plant parasitic nematodes by including piperine or a derivative thereof as an active ingredient, a method for controlling plant parasitic nematodes using the same, a method for controlling plant diseases, and a kit comprising the same .

The present invention provides an insecticidal composition containing piperine or a derivative thereof as an active ingredient.

In addition, the present invention provides a pesticidal method comprising the step of treating a subject in need thereof with a composition containing piperine or a derivative thereof as an active ingredient.

In addition, there is provided a method for controlling or treating a plant disease, comprising the step of treating a subject in need thereof with a composition containing piperine or a derivative thereof as an active ingredient.

In addition, the present invention provides an insecticidal kit against pine wood nematode ( Bursaphelenchus xylophilus ) comprising a composition containing piperine or a derivative thereof as an active ingredient.

In addition, there is provided a kit for controlling or treating pine wilt disease comprising a composition containing piperine or a derivative thereof as an active ingredient.

According to the present invention, the composition containing piperine or a derivative thereof as an active ingredient not only kills the larvae and adults of the plant parasitic nematode, Bursaphelenchus xylophilus , but also inhibits the differentiation of eggs. As it is confirmed to exhibit activity, the composition containing piperine or a derivative thereof as an active ingredient can be provided as an effective insecticidal agent against plant parasitic nematodes and an agent for controlling or treating plant diseases.

1 is a graph confirming the nematicidal effect on pine wood nematode ( Bursaphelenchus xylophilus ) at various concentrations of piperine. The graph shows the mean ± of three replicates, ** indicates P<0.01 compared to the untreated control group, *** indicates P<0.001 compared to the untreated control group.
2 is a graph analyzing the degree of death per hour of the pine wilt nematode ( Bursaphelenchus xylophilus ) according to the presence or absence of piperine or 5-iodoindole (5-iodoindole).
3 is a view confirming the effect of piperine on the pine wilt nematode ( Bursaphelenchus xylophilus ). Figure 3a is a graph evaluating the effect of piperine on the movement (thrashing) of the pine wilt nematode ( Bursaphelenchus xylophilus ) and FIG. 3c is a photograph confirming the effect of piperine on the locomotor traits of the pine wood nematode ( Bursaphelenchus xylophilus ). The graph is shown as the mean ± standard deviation of three repeated experiments, ** indicates P<0.01 compared to the untreated control group, *** indicates P<0.001 compared to the untreated control group.
4 shows carrots ( Daucus carota ) according to piperine treatment concentrations (a: untreated, b: 25 μg/mL, c: 50 μg/mL, d: 100 μg/mL) to evaluate the phytotoxicity of piperine. And Western fern fern ( Raphanus raphanistrum ) It is a photograph evaluating the effect on seed germination.
5 is a diagram illustrating a site where piperine, a piperine derivative, and ivermectin molecularly interact with a GluCL receptor (glutamate gated chloride ion channel receptor; glutamate gated chloride ion channel receptor) in silico .

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written down.

Hereinafter, the present invention will be described in more detail.

The present invention provides an insecticidal composition containing piperine or a derivative thereof as an active ingredient.

The "piperine" is a compound contained in the fruits of pepper tree pepper ( Piper nigrum L.), P. longum , P. officinarum, and P. chusii , and the structural formula is C ₁₇ H ₁₉ NO ₃ , It is represented by the following formula (1).

[Formula 1]

Piperine has been reported to exhibit physiological activities such as antioxidant, anti-mutagenicity, and anticancer action, and is known to play a role in enhancing the bioavailability of drugs such as resveratrol, but has an insecticidal effect that can be used as an insecticide or control agent is not well known about

Alkaloids such as piperine are used as pharmaceuticals in oriental medicine and are considered as strong repellents, and it has been reported that pepper or piperine, an active ingredient thereof, exhibits larvicide activity against Anopheles mosquitoes , Some piperine derivatives have been reported to have insecticidal effects on several different insects.

The "derivative" is a similar compound obtained by chemically changing a part of a certain compound, and a piperine derivative is a compound in which a part of piperine is chemically changed, piperlongumine represented by the following Chemical Formula 2 or It may be a compound such as piperonal represented by the formula (3).

[Formula 2]

[Formula 3]

The "insecticide" means that it can be used as an insecticide that exhibits the effect of removing pests that are harmful to people, livestock, farms and forest trees, where the pests are generally harmful to human life directly or indirectly State means insects, nematodes, or worms, and may include various sanitary pests, agricultural pests or forest pests, and specifically may be plant parasitic nematodes that cause various plant diseases in crops and forest trees. Therefore, the insecticidal composition may be characterized in that it prevents infection of plant parasitic nematodes.

The "plant parasitic nematode" is a nematode that parasitizes on all parts of a plant, such as growth points, leaves, stems, and roots, and ingests cell fluid as nutrients through a globule. It can be divided into endoparasitic, semi-endoparasitic, in which nutrients are absorbed by inserting the head into the tissue. Plant parasitic nematodes do little damage to plants at low densities, but plants infected at high densities suffer fatal damage or die.

The plant parasitic nematode is preferably a pine wood nematode ( Bursaphelenchus xylophilus ), a sweet potato root knot nematode ( Meloidogyne incognita ), a carrot root knot nematode ( Meloidogyne hapla ), a peanut root knot nematode ( Meloidogyne arenaria Amplimerlinius ), Subanguina moxae , rice atrophy nematode ) clavicaudatus ), tobacco nematode ( Tylenchorhynchus claytoni ), apple atrophy nematode ( Tylenchorhynchus nudus ), tea root rot nematode ( Pratylenchus loosi ), perilla root rot nematode ( Pratylenchoides clavicaides ), thistle root nematode ( Pratylenchoides clavicaides ), thistle root nematode ( Pratyhenchoides clavicaides ) Spiral nematode ( Rotylenchus arsenjevi ) , nematode nematode ( Helicotylenchus clarkei ), weeping willow nematode ( Criconemoides komabaensis ), corn scale nematode ( Ogma insulicum ), oak nematode ( Ogma querci ) varionodus ), zelkova bark nematode ( Hemicycliophora epicharoides ), and walnut shell nematode ( Hemicycliophora juglandis ) may be selected from the group consisting of, and more preferably, pine wood nematode ( Bursaphelenchus xylophilus ), but is not limited thereto.

The insecticidal composition is selected from the group consisting of oil sprays, emulsifiable concentrates, wettable powders, fluids, granules, dusts, aerosols, smokers, vaporizable formulations, smoking formulations, toxic attractants, and resin formulations. It can be prepared in a dosage form.

The insecticidal composition may further include a material contained in a commonly used control agent or disinfectant, and in addition to the active ingredient, a pharmaceutically acceptable solid carrier, liquid carrier, liquid diluent, liquefied gas diluent, Solid diluents, or other suitable adjuvants, for example, surfactants such as emulsifying, dispersing or foaming agents may further be included. The control composition in which the active ingredient and the excipient are mixed can be formulated into various formulations known in the agrochemical field, and any formulation method commonly used in the agrochemical field can be used for formulation.

The insecticidal composition can kill the larvae or adults of the plant parasitic nematodes. The “larva” refers to a nematode from hatching from an egg to before becoming an adult, and a nematode that has gone through several larval stages depending on the developmental stage to lay eggs is referred to as an “adult”.

Piperine or a derivative thereof contained in the insecticidal composition may bind to the GluCL receptor (glutamate gated chloride ion channel receptor; glutamate gated chloride ion channel receptor) of the plant parasitic nematode.

The insecticidal composition can prevent the egg hatching of the plant parasitic nematodes, and thus can control the plant parasitic nematodes from being infected by the vector.

Specifically, in the case of the pine wilt nematode ( Bursaphelenchus xylophilus ), it has a life cycle of growing into an adult male and female through eggs, 4 stages of larval stage (J1 ~ J4 stage), 4 molting, and invades the body of the vector at the larval stage. Therefore, the insecticidal composition of the present invention not only inhibits the infection of plant parasitic nematodes in trees, but also blocks the larval stage of plant parasitic nematodes, thereby preventing the spread of plant parasitic nematodes through vectors in plants killed by plant parasitic nematodes. has the effect of

The insecticidal composition may contain the piperine or its derivative in an amount of 10 μg/mL to 100 μg/mL, preferably, it may include piperine or a derivative thereof in an amount of 30 μg/mL to 80 μg/mL, but , but not limited thereto.

In addition, the present invention provides a pesticidal method comprising the step of treating a subject in need thereof with a composition containing piperine or a derivative thereof as an active ingredient.

The subject is a plant that is infected with or not infected with plant parasitic nematodes, and preferably at least one selected from the group consisting of pine, pine, cypress, coastal pine, European pine, and lataeta pine, and more preferably It may be pine, but is not limited thereto.

The insecticidal method may further include the step of treating the insecticidal composition to the egg, its larva, its adult, or its habitat of the plant parasitic nematode.

In addition, the present invention provides a method for controlling or treating a plant disease comprising the step of treating a subject in need thereof with a composition containing piperine or a derivative thereof as an active ingredient.

The "control" refers to any action of suppressing or delaying plant diseases by treating a subject with the composition according to the present invention. The "treatment" refers to any action in which symptoms of plant diseases are improved or beneficially changed by treating a subject with the composition according to the present invention.

In addition, the present invention provides an insecticidal kit against pine wood nematode ( Bursaphelenchus xylophilus ) comprising a composition containing piperine or a derivative thereof as an active ingredient. In addition, the present invention provides a kit for controlling or treating pine wilt disease comprising a composition containing piperine or a derivative thereof as an active ingredient.

The "kit" means, for example, to independently form an insecticidal composition in the form of particles, granules or pellets, and to prepare it in a form usable at the same time.

Hereinafter, examples will be described in detail to help the understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

The following examples were repeated, and the results are presented as mean ± standard deviation, and statistical analysis using one-way ANOVA according to the SPSS version 23 (SPSS Inc., Chicago, IL, USA) Dunnett test was performed, and the p value was <0.05, <0.01, or <0.0001 was considered significant.

Example 1. Evaluation of the effect of piperine on the survival rate of pine wilt nematodes

In order to evaluate the insecticidal effect of piperine as a natural insecticide on pine wilt nematodes, the survival rate of pine wilt nematodes according to the treatment of piperine and piperine derivatives was confirmed.

In a 96-well plate, 200 pine wilt nematode larvae to be evaluated for insecticidal effect were prepared per plate well, and piperine, piperine derivatives piperlongumine or piperonal were added at 0 μg/mL and 5 μg, respectively. /mL, 10㎍/mL, 30㎍/mL, and 50㎍/mL, and after incubation for 24 hours at 22 ℃, UV LED illumination and iRiS™ Digital Cell Imaging System (iRiS™ Digital Cell Imaging System; Logos Bio Systems, Korea) was counted with an optical microscope equipped with the number of living pine wilt nematode larvae and converted to a percentage compared to the untreated group (Fig. 1).

As shown in FIG. 1, piperine showed an insecticidal effect on pine wilt nematodes, and 40% of pine wilt nematode larvae were killed in the piperine 30 μg/mL treatment group, and 90% in the piperine 50 μg/mL treatment group. % of pine wilt nematode larvae were killed, and it was confirmed that piperine had an excellent insecticidal effect against pine wilt nematodes.

In addition, in the piperine 50 μg/mL treatment group, it was confirmed that all certain larvae (J2 stage) lost the activity of Pine wilt nematode larvae (J2 stage) within 1 hour of treatment with piperine, and were completely killed within 6 hours (Fig. 2).

As shown in Figure 2, the positive control group, 5-iodoindole (5-iodoindole) treatment group showed a general killing pattern of pine wilt nematodes, whereas in the piperine treatment group, curling reaction and aggregation A unique insecticidal pattern such as Referring to the previous studies, the unique insecticidal pattern of piperine against the pine wilt nematode as described above suggests that it can be used as an excellent insecticide that kills the nematode in a short period of time.

Example 2. Evaluation of the effect of piperine on the egg hatching rate of pine wilt nematodes

A strong insecticide or control agent not only kills the target pest, but is accompanied by the effect of inhibiting the egg hatching of the pest. In a previous study, halogenated compounds such as 5-iodoindole inhibited egg hatching of nematodes and showed an insecticidal effect.

In order to evaluate whether piperine as an insecticide or control agent inhibits egg hatching of pine wilt nematodes, the egg hatching rate was checked by treating pine wilt nematode eggs with piperine.

In a 96-well plate, the eggs of pine wood nematodes were concentrated in plate wells using the egg settling method, washed repeatedly with sterile water, and counted. Piperine was treated with 0 μg/mL, 30 μg/mL and 50 μg/mL, and the treated plate was incubated at 22° C. for 24 hours to promote egg hatching of Pine wilt nematodes, and then hatched larvae using an optical microscope. And the number of eggs that did not hatch were counted. All experiments were repeated twice, the results were presented as mean ± standard error, and the percentage hatch rates (HR) of pine wilt nematode eggs were calculated as follows:

HR(%) = [larva / (egg + larva) Х 100].

As shown in FIG. 3b , pine wilt nematode eggs treated with piperine at 30 μg/mL or 50 μg/mL for 24 hours did not hatch. In particular, in the group treated with piperine 50 μg/mL, embryo development was impaired in J1 and J2 eggs, and hatching was inhibited by about 90%, and the egg hatching rate of Pine wilt nematodes showed a tendency to decrease depending on the concentration of piperine treatment.

Example 3. Evaluation of the effect of piperine on the motility of pine wilt nematodes

The nematode motility in the liquid medium moves in a standardized flexible motion, and the thrashing behavior, which is defined as moving the head from one direction to another, is a characteristic that evaluates the motility of nematode larvae.

In order to evaluate the effect of piperine on the motility of pine wilt nematodes, the number of thrashing behaviors and movement patterns were evaluated in pine wilt nematode larvae exposed to piperine.

After exposing P. nematode larvae to a liquid medium containing piperine for 24 hours, they were scanned for 1 minute under an optical microscope equipped with an iRiS™ Digital Cell Imaging System (Logos Bio Systems, Korea) at 20X magnification. Thrashing behaviors were counted and movement patterns were image monitored at 20X magnification. Pine wilt nematode larvae were not used for analysis if they were abnormally coiled or showed no movement.

As shown in FIG. 3A , the frequency of thrashing behavior of pine wilt nematode larvae decreased with piperine treatment. In the piperine 50 μg/mL treatment group, the thrashing frequency was significantly reduced, and the thrashing behavior of the surviving pine wilt nematodes appeared slowly. Figure 3c is a photograph of the thrashing behavior of pine wilt nematode larvae in the piperine 50 μg/mL treatment group and the control group at 20X magnification under an optical microscope, and in the control group untreated with piperine, the pine wilt nematode larvae On the other hand, it was found that the thrashing behavior of pine wilt nematode larvae was suppressed in the piperine-treated group.

Considering the above results, it is suggested that piperine has an overall effect of inhibiting the movement and muscle function of pine wilt nematodes.

Example 4. Toxicity assessment of piperine on plants

To determine whether piperine is toxic to plants, the effect of piperine on plant seed germination was evaluated in a seed germination experiment.

Seeds of carrot ( Daucus carota ) and fern ( Raphanus raphanistrum ) were soaked in sterile water for one day, washed with 1 mL of 100% ethanol, and sterilized in 3% sodium hypochlorite solution for 15 minutes. did. The seeds were then sterilized and washed several times, and Murashige & Skoog medium (0.86 g/mL L) sterilized seeds were cultured at 25° C. for 2 days to evaluate the degree of germination.

As shown in FIG. 4 , both seeds of carrot ( Daucus carota ) and fern fern ( Raphanus raphanistrum ) germinated in the piperine-treated group after 2 days, and it was confirmed that piperine was not toxic to plants.

Example 5. Molecular effect of piperine on nematodes ( in silico )

Ivermectin, an insecticide for nematodes, exhibits an insecticidal effect by inducing nerve paralysis by acting on GluCL receptors (glutamate gated chloride ion channel receptor) involved in nerve signal transduction of nematodes. In order to determine whether piperine exhibits an insecticidal effect through a mechanism similar to that of ivermectin, the interaction between piperine and GluCL receptor was molecularly analyzed using molecular docking technology.

The GluCL receptor of Caenorhabditis elegan was used to analyze the binding form with piperine, and to identify the GluCL receptor active site, it was compared with the GluCL receptor binding form of ivermectin or piperlongumin, a derivative of piperine.

GluCL receptors were prepared using the Protein Preparation Wizard of Schrodinger software package, and ligands were prepared using LigPrep software. Docking was performed using Schrodinger software 11.4 (Schrodinger Software Solutions, USA) in ultra-precision (XP) flexibility mode.

It has been shown that ivermectin binds to the transmembrane domain, that is, the active site, of the Chain D/E interface of the GluCL receptor, and when ivermectin is removed from the GluCL receptor-ligand complex, a grid with a cubic size of 18 Å × 18 Å × 18 Å was formed As shown in FIG. 5 , ivermectin interacted with Glu at position 219 of the GluCL receptor. The 219th Glu of the GluCL receptor interacts with a ligand and is a major site for activation of the GluCL receptor.

Referring to FIG. 5 , piperine was found to interact with Glu at position 219 of the GluCL receptor similarly to ivermecti.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. Do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims (19)

As a composition for insecticidal pine wood nematode ( Bursaphelenchus xylophilus ) containing piperine as an active ingredient,
The insecticidal composition is an insecticidal composition, characterized in that it contains the piperine in an amount of 10 μg/mL to 100 μg/mL. According to claim 1, wherein the insecticidal composition is a pine wood nematode ( Bursaphelenchus xylophilus ) Insecticidal composition, characterized in that to prevent infection. delete delete The composition of claim 1 , wherein the insecticidal composition is an oil spray, emulsifiable concentrate, wettable powder, fluid, granule, dust, aerosol, fumigant, vaporizable formulation, smoking formulation, toxic attractant, and resin formulation. Insecticidal composition, characterized in that the formulation selected from the group consisting of. According to claim 1, wherein the insecticidal composition is an insecticidal composition characterized in that to kill the larvae or adults of the pine wood nematode ( Bursaphelenchus xylophilus ). According to claim 6, wherein piperine contained in the insecticidal composition is GluCL receptor (glutamate gated chloride ion channel receptor; glutamate gated chloride ion channel receptor) of the pine wood nematode ( Bursaphelenchus xylophilus ) Insecticide characterized in that it binds to composition. According to claim 1, wherein the insecticidal composition is an insecticidal composition for preventing the egg hatching of the pine wood nematode ( Bursaphelenchus xylophilus ). delete As an insecticidal method of pine wood nematode ( Bursaphelenchus xylophilus ) comprising the step of treating a subject in need thereof with a composition containing piperine as an active ingredient,
The composition is an insecticidal method, characterized in that it comprises the piperine in an amount of 10 μg/mL to 100 μg/mL. The method according to claim 10, wherein the subject is a plant infected with the pine nematode ( Bursaphelenchus xylophilus ). The method according to claim 11, wherein the plant is one or more selected from the group consisting of pine, pine, pine, coastal pine, European pine, and rataeta pine. According to claim 11, wherein the insecticidal method further comprises the step of treating the insecticidal composition to the eggs, larvae, adults thereof, or their habitat of the pine wilt nematode ( Bursaphelenchus xylophilus ). delete A method for controlling or treating pine wilt disease, comprising the step of treating the composition of claim 1 to a subject in need thereof to insecticidal pine wilt nematode ( Bursaphelenchus xylophilus ). delete delete delete delete

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